Nowadays mass spectrometric analysis of mixtures, particularly of mixtures of macromolecular substances, for example large biomolecules, generally uses separation methods in liquid phase and ionization of the substances, which are at least partially separated in this way, by electrospray ionization in order to generate the analyte ions. Electrospray ionization is an extremely soft ionization, however, which essentially provides only molecular ions without fragment ions, and hence only results in a determination of the molecular weight of the substances. To be more precise, pseudo-molecular ions are formed. These are protonated or deprotonated ions which differ from the true molecular ions by the weights of the excess or missing protons. Alternative ionization methods such as laser ionization also produce ions which form ions from neutral substances by the addition or removal of protons. In the following, the spectra of pseudo-molecular ions will simply be called “molecular spectra”, and “molecular ions” will always be used to describe the pseudo-molecular ions created by adding or removing electrically charged units (protons, and in some cases also alkali ions).
For the best possible characterization and identification of the substances, however, further knowledge is required in addition to the molecular weight, especially information concerning the structure; for proteins, this means information concerning partial sequences of the amino acids. The partial sequences can be determined from spectra of molecular ions which have been fragmented by a suitable method. Other characteristic properties of the substances are obtained by acquiring mass spectra, where the substances or their molecular ions have been chemically or physically modified.
Automated methods for measuring the spectra of fragment ions are usually used to gain information about the structure. Measurement of the fragment ions requires special “tandem mass spectrometers” (often abbreviated to MS/MS), in which fragmentation of suitable, preferably multiply charged, molecular ions is possible; the ions have to be selected beforehand by a mass spectrometric filter. Spectra of this type are frequently called daughter ion spectra; in some mass spectrometers it is possible to select, fragment and measure daughter ions again. One then obtains granddaughter ion spectra. A variety of methods for fragmenting the substances have been elucidated, including fragmentation using high-energy collisions with neutral particles (collision gas), fragmentation by absorption of energy from incident photons, usually in the infrared (IRMPD=infrared multi photon dissociation), and fragmentation resulting from reactions with electrons, negative ions or highly excited neutral particles.
Certain substances can also form ions of a type other than fragment ions if their ions react with other particle. Thus, complex formations with alkali ions, with metal ions or with molecules of solvents (salvation) are sometimes characteristic of certain types of compounds. Their spectra can also be acquired alternately to molecular ions. For example, a method has been published whereby lithium salts are added in the spray capillary to generate lithium ion complexes of substances which are normally difficult to ionize in electrospray ion sources. Reactions of mixtures of multiply positively charged biomolecules with negative ions make it possible to utilize a process called “charge stripping” to produce mixtures of singly charged ions, which are much easier to interpret. The spectra of such ions which are generated in a chemical or physical reaction are grouped together below as “special mass spectra”.
In the automatic MS/MS methods mentioned, spectra of molecular ions and fragment ions are acquired in turn. This requires information concerning the occurrence of newly appearing substances, and this information has to be obtained immediately and very rapidly from the molecular spectra just measured. The alternate acquisition of molecular spectra and fragment spectra and the lack of information concerning the concentrations of newly occurring substances to be expected in the future make it impossible to optimize this method, since a decision must be made immediately, using the molecular spectra, as to which molecular ions are to be selected and fragmented. If, despite the temporal separation, the flow of substances contains several overlapping substances (which is practically always the case in complex mixtures), then the decision on a substance can be very difficult because, at best, information about the beginning of a substance batch is available, but there is no complete profile of the substance batch with position and height of the maximum. The substance batches from the separating device (often called “substance peaks”) can certainly have a very different concentration and demonstrate complex overlapping patterns. Despite using intelligent algorithms, substances disappear from the substance flow before the spectra of the fragment ions can be scanned. On the other hand, such fragment ion spectra are frequently taken too early, far before the substance has reached its maximum concentration; their quality is then frequently too poor for further processing. There is usually not enough time for a repeat scan, because new substance batches are already appearing.
Depending on the concentration of the substances supplied, scanning the molecular spectra takes only a few tenths of a second in modern mass spectrometers; scanning daughter ion spectra usually takes several times as long. Nevertheless, it is possible to scan between one and five pairs of molecular ion and daughter ion spectra per second.
Conventional liquid chromatography provides substance batches whose profiles can quite easily have a width of roughly between five and thirty seconds. A conventional, automatic scan of daughter ion spectra is quite promising here. However, modern separation methods have ever larger separation selectivities and, associated with this, ever shorter temporal widths of the substance batches separated. The use of very fine capillaries in the so-called nano-LC already shortens the time in which a substance is delivered to a few seconds. In capillary electrophoresis, it is possible to achieve profile widths of the substance batches of between one and three seconds. In electrophoretically mediated capillary chromatography the batch widths are already less than one second. Chip-based micro-separation systems generate substance batch widths of only a few tenths of a second. For separation systems in which the substance mixtures already change rapidly within tenths of a second, an alternate measurement of molecular spectra and daughter ion spectra can no longer be used because of the concentration changes in the time shift between the two measurements, even if the concentration is very high in the substance batches themselves, which makes it possible to have a very short scanning time for the mass spectra.